Neural stem cells (NSCs) play critical roles in generating neurons and glia during both embryonic development and adult life. Aberrant NSC behavior could lead to neurological disorders, especially those with developmental etiology, and brain tumors. Drosophila neuroblast provides an excellent model for understanding fundamental aspects of NSC regulation in vivo. The long-term goal of this proposal is to elucidate mechanisms regulating the self-renewal and differentiation of NSCs through combined cell biological, genetic, and molecular analyses. Drosophila neuroblasts undergo stem cell-like self-renewing asymmetric divisions. In each division, one daughter cell remains as a stem cell while the other is committed to differentiation. Factors controlling stem cell self-renewal and differentiation are distributed in a polarized fashion and the mitotic spindles are oriented with respect to this polarity axis, ensuring proper segregation of these important factors between the two daughter cells. Numb is such a factor that is segregated to the differentiating daughter cell during neuroblast division, where it inhibits the renewal of neuroblast cell fate. The asymmetric segregation of Numb requires Partner of Numb (Pon). In the last four years we have focused on identifying molecules that impinge on Pon to regulate Numb and those that regulate mitotic spindle orientation. We have learned that a number of mitotic kinases directly act on Pon and Numb. This raises the interesting possibility that during NSC asymmetric division, a series of mitotic kinases act in concert to ensure the faithful segregation of key cell fate determinants to the appropriate daughter cells. We propose to test this hypothesis. We have also learned that a number of tumor suppressors and kinases are involved in regulating mitotic spindle orientation. We propose to investigate the biochemical and genetic relationships between these signaling molecules and to place them into a pathway. We also propose to assess the contribution of the Pon/Numb pathway and the spindle orientation pathway in controlling NSC self-renewal and to identify new players involved in this process. Given the evolutionary conservation of the molecules studied in this proposal and the conservation of mechanisms underlying NSC asymmetric division, knowledge to be gained from this study shall provide important insights into the understanding of mammalian NSC regulation in vivo and ultimately treating diseases originated from dysfunctional NSCs. PUBLIC HEALTH REVELANCE: Neural stem cells are multi-potent cells that generate the diverse cell types in our brain. The goal of this proposal is to achieve a mechanistic understanding of the basic properties of these special cells and how disease may arise when neural stem cells behave abnormally. Accomplishment of the proposed aims will ultimately help understand and treat a number of neurological disorders originated from aberrant neural stem cells, such as neurodegenerative diseases and brain tumors.